This invention is the culmination of a lengthy research endeavor that began with an observation that a particular group of immunodeficient mice, inoculated with Kaposi's sarcoma (KS) cells, did not develop KS as expected. The researchers extracted KS cells from AIDS patients, grew the cells in vitro and then injected the cells into the immunodeficient mice, expecting the mice to develop KS. While most mice injected with the cells did develop KS, the researchers surprisingly identified a group of ten mice in which KS failed to develop.
In attempting to explain the failure of these mice to develop KS, the researchers first considered whether the anomaly was the result of a laboratory error, such as a failure to properly inject the mice. Closer observation revealed that all ten KS-negative mice were pregnant. A group of females and males had been mistakenly housed together. While the male mice had grown large malignant tumors, the females had only small tumors or no tumors at all. Further observation indicated that females with small tumors were in later stages of pregnancy, and those with no tumors were in early stages of pregnancy. Based on these collective observations, the researchers surmised that one or more factors produced in the early stages of pregnancy was responsible for impeding the development of KS lesions.
In the ensuing months, the group designed and executed a controlled study of the effects of pregnancy on KS. The results of the study were published in the May 4, 1995, issue of Nature.1 In this study, the researchers injected KS cells into 24 female mice and 21 male mice. Four of the female mice were then caged with 2 males. All 4 females became pregnant. One month later, the males had developed large malignant tumors; and as expected, females in late stages of their pregnancy had developed smaller tumors, while newly pregnant females were tumor free.
These results confirmed the group's suspicions that a pregnancy-related factor was responsible for the inhibition of KS. The group noted that the pattern of no KS lesions in early stages of pregnancy and increasingly larger lesions at later stages of pregnancy inversely corresponds to the production of human chorionic gonadotropin (hCG). High levels of hCG are produced during early pregnancy, while lower levels are produced in later stages of pregnancy. The researchers investigated the anti-KS activity of crude commercial hCG preparations. These investigations revealed that some lots of some commercially available crude hCG preparations exhibited the same anti-KS effect seen in the pregnant mice.
In a related study published in the Oct. 24, 1996 issue of The New England Journal of Medicine,2 the investigators tested hCG preparations on 30 human subjects. The results were compared with 6 others who were given a placebo. Twenty-four subjects received intralesional injections of preparations of hCG at doses of 250, 500, 1000, or 2000 international units (IU) three times a week for 2 weeks. In 5 of the 6 subjects who received the 2000 IU hCG dose, KS lesions shrank significantly or completely disappeared. Lesions in at least one subject in each of the 250, 500, and 1000 dose ranges also shrank or disappeared. This study provided further evidence that some lots of some commercial hCG preparations exhibit anti-KS effects. The results also demonstrated that certain crude commercial preparations of hCG can reduce or reverse symptoms of KS in humans.
In another related experiment, the researchers administered a 2000 IU hCG dose preparation to 6 subjects. These subjects were compared to six others who received a placebo. KS lesions shrank or completely disappeared in all 6 subjects receiving hCG, while none of the controls showed any change.
These results were further confirmed in a study published in the January/February 1998 issue of the Journal of Human Virology.3 In this study, 13 subjects with advanced AIDS-KS and visceral KS were treated with a commercial hCG preparation known to have anti-KS effects. Of 12 subjects treated with the hCG preparation, 5 had a dramatic response to the therapy and overall tolerance to the treatment was excellent.
In addition to the surprising anti-KS properties revealed by this series of experiments, the group also observed that the hCG preparations had no toxic side effects, common in other anti-cancer therapeutics. In fact, some subjects reported increased energy, enhanced appetite and even weight gain, while the control group showed no such changes.
The variability of results among lots and sources of the hCG preparations led the group to conclude that the active factor, which was clearly present only in some lots from some sources, could not be hCG itself. And although the crude preparations produced some positive results, the variability in the crude preparations was not conducive to the development of a standard therapeutic protocol. The variability of results prevented an accurate determination of ideal dose, route or frequency of administration.
The group had made following observations:                even though the commercial preparations of hCG had a standardized amount of hCG, the observed activity varied from manufacturer to manufacturer and within a particular manufacturer's hCG preparations;        some of the commercial hCG preparations had no activity;        pure hCG (native purified and recombinant) had none of the observed activities;        when some commercial preparations of hCG were intentionally depleted of hCG activity, they maintained the previously observed activities; and        mice do not have the genes for chorionic gonadotropin, but the observed activities were found in sera and the urine of mice in early pregnancy.        
These observations led to the hypothesis that the activity of the hCG preparations is mediated by one or more molecules which co-purify with hCG in only some of the commercial preparations.
Next, the researchers tested fragments of hCG for therapeutic activity. A group of fragments from the β-chain of hCG (SEQ ID NO: 1) were identified which exhibited various anti-HIV, anti-cancer and pro-hematopoietic effects.4 Among the peptides exhibiting such effects were the Satellin A1 peptide [β-hCG 45-57: Leu-Gln-Gly-Val-Leu-Pro-Ala-Leu-Pro-Gln-Val-Val-Cys (SEQ ID NO: 18); the Satellin A1 branched peptide: β-hCG 45-57 [Leu-Gln-Dab(Pro)-Val-Leu-Pro-Dab(Pro)-Leu-Pro-Gln-Val-Val-Cys (see SEQ ID NO: 18, for primary sequence), where “Dab” represents diaminobutyric acid, and Dab(Pro) indicates a proline peptide-bonded to the amino side chain of Dab], the Satellin A2 circularized peptide [β-hCG 45-57 with a cysteine residue added to the N-terminus, circularized via a disulfide bond between the cysteine residues: Cys-Leu-Gln-Gly-Val-Leu-Pro-Ala-Leu-Pro-Gln-Val-Val-Cys (SEQ ID NO: 19)], and the Satellin B peptide [β-hCG 109-119: Thr-Cys-Asp-Asp-Pro-Arg-Phe-Gln-Asp-Ser-Ser (SEQ ID NO: 22). Other β-hCG (SEQ ID NO: 1) peptide fragments demonstrating some degree of efficacy included the following: 109-145, 47-55, 48-56, 45-57 fused to 109-119, and 45-57 in combination with 109-119.
Although the already-discovered group of β-hCG fragments clearly were efficacious in treating KS and other conditions, it appeared that one or more factors remained to be discovered, i.e., the factor or factors which accounted for the higher degree of efficacy exhibited by the commercial hCG preparations. As discussed more fully in the detailed description of the invention, the present inventors have isolated two such highly active factors. The inventors have named these peptides “Maternin™” for the purpose of future commercialization, and the peptides are generically referred to herein as the MA peptides: MA (SEQ ID NO: 2) and pMA (SEQ ID NO: 3), which are the subject of the instant application.
Other researchers have investigated the use of preparations of heterodimeric hCG in the treatment of HIV. Such preparations have been shown to reduce the reverse transcriptase activity in HIV-1 infected lymphocytes and monocytes in culture5 and to prevent transmission of HIV from lymphocytes to trophoblasts in vitro.6 One researcher has proposed the use of doses of hCG below those necessary to induce a humoral immune response for treating of HIV infection.7 Researchers have also reported that treatment with hCG improves T cell counts and physical symptoms in certain HIV infected subjects.8 However, none of these researchers have successfully isolated novel, highly active peptides from commercial preparations of hCG or urine.
Additionally, the β-hCG (SEQ ID NO: 1) has been reported to reduce HIV production in lymphocytes at doses from 100 pg/ml to 100 μg/ml and in monocytes at doses up to approximately 10 μg/ml, with higher doses actually increasing the level of viral production in monocytes.9 Lunardi-Iskandar et al.10 reported that hCG, β-hCG (SEQ ID NO: 1), as well as a β-hCG carboxy-terminal peptides of amino acids 109-145 and 109-119 are efficacious in the treatment of Kaposi's Sarcoma. However, this work did not contemplate the presence of additional specific β-hCG-derived products with highly active anti-KS properties, or any of the other therapeutic effects for such products as described herein.
The inventors also previously identified fractions of early pregnancy urine with consistently potent therapeutic effects, such as anti-HIV, anti cancer, anti-wasting and pro-hematopoietic effects.11 These effects were shown to be present in fractions of a source of native hCG or β-hCG. Particularly active fractions included material eluting from a gel filtration column with an apparent molecular weight of approximately 40 kD, 15 kD or 2-3 kD. The same peak fractions not only killed KS tumor cells but also inhibited HIV-1 replication in vitro, suppressed HIV-1 expression in HIV-1 transgenic mice, and inhibited the development of AIDS in SIVMAC 251 infected macaques.12 The instant invention is an extension of this work, and is the result of a lengthy and extensive program of experimentation directed towards identifying the one or more pharmacologically active constituents of early pregnancy urine.
Following our original report, several groups attempted to identify one or another of the molecules responsible for these activities. These reports were limited to in vitro studies showing some inhibition of KS cell growth in culture or of decreased HIV replication with some already well-known urinary products such as EDN RNase,13 other RNases,14 lysozyme,15 and the native gylcosylated β-core of β-hCG16 or with a putative purified hCG.17 One group described some in vitro anti-KS activity with a low molecular weight fraction obtained from a crude hCG preparation, the chemical nature of which was not defined or further characterized.18 Importantly, none of these reports showed in vivo results, and none showed the multiple effects described here. Moreover, with the exception of the hCG related products, none of these molecules are known to be relatively high during early pregnancy, and the report of positive results with native glycosylated β-core emphasized selective inhibition only of the growth of KS cells.19 
In our earlier work20 and confirmed in this report, we found no activity in vitro or in vivo in any of the various experimental systems described here with purified native glycosylated β-core, β-hCG, α-hCG, and/or hCG. Earlier and distinct from these in vitro studies, Russo and co-workers reported that a crude commercial hCG preparation (ProFasi, Organon) inhibited carcinogenesis induced by DMBA in rats.21 As indicated, we have demonstrated that pure hCG has no effect on any tumor cell we have studied, and the crude ProFasi hCG was inactive.